Effects of different live feed on ballan wrasse (Labrus bergylta) larval hepatocyte and enterocyte development

Transcription

1 Effects of different live feed on lln wrsse (Lrus ergylt) lrvl heptocyte nd enterocyte development Mrte Romundstd Mrine Costl Development Sumission dte: Jnur 2015 Supervisor: Elin Kjørsvik, IBI Co-supervisor: Jn-Ove Evjemo, IBI Norwegin University of Science nd Technology Deprtment of Biology

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3 Acknowledgements This mster thesis ws written t the Deprtment of Biology, t the Norwegin University of Science nd Technology (NTNU), under the guidnce of Professor Elin Kjørsvik t the Deprtment of Biology NTNU nd resercher Jn Ove Evjemo NTNU. The experimentl work ws crried out t the NTNU Centre of Fisheries nd Aquculture (Sel) nd in the lortories of SINTEF Fisheries nd Aquculture. This experiment ws prt of the reserch project Produksjon v erggylte (900554), funded y the Fishery nd Aquculture Industry Reserch Fund (FHF), collortion etween NTNU, SINTEF, Nofim, NIFES nd the Institute of Mrine Reserch, s well s lln wrsse juvenile nd slmon producers. I would like to thnk Elin Kjørsvik for her guidnce nd ptients in writing this thesis, Jn Ove Evjemo for the help nd guidnce in the first feeding experiment nd Tor Brdl for invlule help nd ssistnce in the l. I would lso like to thnk Od Skognes Høylnd nd Mri Stvrkki for gret collortion in the first feeding experiment, nd to Mren Rnheim Ggnt, Stine Wiorg Dhle nd Andres Hgemnn for ll the help with fish feeding nd live feed rering. Thnks to LAKS nd ll fellow students t Sel for gret converstions, fun prties nd good support during these lst two yers. I would lso like to thnk my fmily nd my oyfriend for ll their love nd support through the mster thesis process. Trondheim, Jnury 2015 Mrte Romundstd I

4 Astrct Slmon lice infesttions re one of the iggest chllenges for Norwegin slmon frming tody. Delousing gents hve een the solution to these infesttions in the pst, ut overuse nd wrong dosges hve cused the lice to ecome resistnt to the chemicls nd thus rendering them ineffective. As result clener fish hve een proposed s n ll-nturl delousing gent. The lln wrsse hs een proposed s the idel clener fish for Norwegin wters nd is eing cught in the wild nd put in the fish pens with the slmon. To e le to use lln wrsse on lrger scle nd in sustinle wy, it hs een proposed to egin frming of the lln wrsse. This will secure stle supply of this fish to the slmon frms. One of the keys to successfully rering lln wrsse is to figure out the idel feeding regime in the lrvl stge of development when the lrve re dependent on live feed. The trditionl live feed of rotifers nd rtemi do not meet the nutritionl requirements of mrine fish lrve nd hs to e enriched to meet these requirements. Copepods hs een suggested s more suitle live feed s it is the nturl prey of most wild mrine fish nd meets their nutritionl requirements thus not needing to e enriched. In this study the trditionl live feed of rotifers nd rtemi ws compred with using intensively rered copepods (Acrti tons) exclusively through the whole live feed period of lln wrsse lrve. The Rotifer-group ws fed enriched rotifers from 4 dph until 30 dph, nd ws co-fed enriched rotifers nd enriched rtemi etween 30 dph nd 40 dph. From 40 dph until the end of the experiment (45 dph) the Rotifer-group ws fed enriched rtemi exclusively. The Copepod-group ws fed intensively rered copepods exclusively throughout the experiment (4-45 dph). The lln wrsse lrve were fed Acrti tons nuplii until 15 dph, then s the lrve grew they were fed copepodits from 15 dph until 30 dph. From 30 dph to 45 dph the lrve were fed comintion of copepodits nd dult Acrti tons in 2:1 rtio. The two groups were compred to see if the nutritionl differences of the live prey hd n effect on different growth prmeters nd how it ffected the gut nd liver development in the lln wrsse lrve. The results from this study indictes tht intensively rered Acrti tons ws etter live feed for lln wrsse lrve compred to enriched rotifers nd enriched rtemi. The lrve tht were fed copepods showed etter growth nd hd etter liver nd gut development thn the lrve fed enriched rotifers nd rtemi. II

5 Contents 1 Introduction Bckground Slmon lice nd clener fish Blln wrsse nd live feed Gut nd liver development Aim of the study.4 2 Mterils nd methods First feeding experiment Live feed production Cultivtion nd hrvesting of microlge (Rhodomons ltic) Cultivtion of copepods (Acrti tons) Egg production Production of copepod nuplii for feed Cultivtion, enrichment nd hrvesting of rotifers (Brchionus sp.) Cultivtion of Artemi frnciscn Lrvl smpling nd fixtion Stndrd length nd myotome height Dry weight nd dily weight increse Survivl Histologicl nlysis Light microscope Trnsmission electron microscope Sttisticl nlysis 12 3 Results Growth nd survivl Dry weight nd dily weight increse Stndrd length (SL) nd myotome height (MH) Survivl Liver morphology Light microscopy Trnsmission electron microscopy Surfce re frction of heptocyte components Are size of nucleus nd mitochondri Mitochondril chrcteristics Gut morphology Light microscopy Trnsmission electron microscopy Surfce re frctions of enterocyte components Are size of nucleus nd mitochondri Mitochondril ppernce.37 III

6 3.3.6 Microvilli density nd height Discussion Lrvl growth nd development Effects of different live feed on lrvl heptocyte nd Enterocyte development Nuclei size nd ppernce Mitochondril development Endoplsmic reticulum Lipid nd glycogen storge Conclusion.46 References.47 Appendix 1.52 Emedding lln wrsse lrve in EPON 52 IV

7 1. Introduction 1.1 Bckground With the rising numer of people on the plnet, food production hs to e expnded to meet the future requirements nd quculture is sid to ply n importnt role is this development. Aquculture, especilly of slmon, hs ecome n importnt industry in Norwy. With production of 1.16 million tons of slmon in 2013 (Norwegin Directorte of Fisheries, 2014), it hs ecome n importnt industry in Norwy. The export of slmon from Norwy ws vlued t 40 illion NOK (Sttistics Norwy, 2014) mking it n importnt export. The slmon industry hs developed lot since the eginning in the 1970`s. The technologicl progress hs mde lrge scle fish frms possiility nd tody one fish frm cn contin fish (Trnger et l., 2014). Although these intensive fish frms re highly successful there re severl chllenges with high density fish rering. The spreding of diseses, the escpe of frmed slmon, which interfere with the wild slmon popultion, nd incresed numers of slmon lice in the Norwegin fjords, re some of the chllenges the industry fces tody (Trnger et l., 2014). 1.2 Slmon lice nd clener fish The slmon louse is n ectoprsite (Kt 1979) tht feeds on the skin of its host (Pike & Wdsworth, 2000). This dmges the skin of the slmon nd cuse lcertions tht cn ecome ftl, especilly when slmon is infected with lrge numer of lice (Grimnes & Jkosen, 1996). In ddition they cn infect the wild slmon nd cuse prolems for the wild popultions (Bjørn et l., 2001; Butler, 2002). The industry hs tried to comt this prolem for long time nd in the pst the min solution hs een to use delousing gents to remove the lice from the slmon. Overuse nd wrong dosges hve cused the lice to ecome resistnt to severl kinds of delousing remedies (Jones et l., 1992; Sevtdl & Horserg, 2003; Lees et l., 2008) nd it is now hrd to effectively get rid of the lice using just chemicls. A solution hs een to use wrsse nd other species of clener fish for delousing the frmed slmon. They cn e put into the pens with the slmon nd will et the lice off the fish. Wrsse hs een used s clener fish since the lte 1980`s (Espelnd et l., 2010), ut hs not een widely used until lter yers. In Norwegin wters the species tht is considered to e the most suited for this purpose is the lln wrsse. It hs ig ppetite, it will effectively clen the lice off the slmon (Ottesen et l., 2008), nd it is ctive t lower tempertures thn 1

8 other wrsse species (Kvenseth & Øien, 2009). As the chemicl delousing gents gets less nd less effective the more one hs to rely on the nturl delousing effects of the wrsse. Most of the wrsse used in Norwegin fish frms tody, re cught in the wild (Kvenseth et l., 2003), ut in longer perspective one hs to think of more sustinle wys of supplying the wrsse to the frms. Frming of lln wrsse would help relieve the pressure on the wild popultions nd help ensure stle supplies of wrsse to the slmon frms. 1.3 Blln wrsse nd live feed. When strting to frm new species there is severl chllenges nd needs for knowledge. How do we get the fish to reed in cptivity, wht is the est htching conditions, how ig re the lrve when they htch etc. One of the iggest chllenges with mrine fish lrve is their generl smll size t htching. The slmon is so developed t htching tht it cn e fed with formulted feed right wy, ut the smller lln wrsse is not s developed t htching nd therefore hs to e fed live feed (Conceico et l., 2010). The trditionl live feed regime used on severl mrine species, consists of first period of feeding with rotifers, nd s the lrve grows igger, they re wened on to the lrger rtemi, efore they re ig enough to e wened on to formulted feed (Southgte, 2003; Conceio et l., 2010). One of the chllenges with these species s food for the fish lrve is tht they do not nturlly meet the lrve s requirements for essentil ftty cids (EFAs). Therefore they hve to e enriched efore they re fed to the lrve (Evjemo & Olsen, 1997; Conceico et l., 2010). EFAs like eicospentenoic cid (20:5n-3; EPA), docoshexenoic cid (22:6n-3; DHA) nd rchidonic cid (20:4n-6; ARA) is sid to e prticulrly importnt for the growth nd development of mrine fish lrve (Srgent et l., 1999; Bell et l., 2003). Not only is the mount of these ftty cids importnt, ut the rtio etween them re lso importnt, the optiml rtio will vry from species to species (Srgent et l., 1999). EFAs stored in the polr lipid frction is sid to e more esily ville for the fish thn ftty cids stored in the neutrl lipids (Izquierdo et l., 2000; Gisert et l., 2005; Wold et l., 2009). When the rotifers nd rtemi re enriched, the essentil ftty cids re stored in the neutrl lipid frction (Rinuzzo et l., 1994; Rinuzzo et l., 1994; Nerhus, 2007) nd re therefore less ville for the developing lrve (Izquierdo et l., 2000; Kjørsvik et l., 2009; Wold et l., 2009). As n lterntive to the more trditionl live feed regime it hs een proposed to feed the lrve with copepods. Both nturlly hrvested nd intensively rered copepods hve een proven to e good live prey option for mrine fish lrve. When compred to feeding with rotifers nd rtemi, copepods s live feed hs resulted in etter growth, survivl nd pigmenttion 2

9 (Evjemo & Olsen, 1997; Luizi et l., 1999; Shields et l., 1999; Imslnd et l., 2006; Eidsvik, 2010). Copepods re the nturl pry of mny wild mrine fish lrve (Hunter, 1981) nd re more suited to meet the nutritionl requirements of the fish lrve. They do not need to e enriched s the mount nd rtio of essentil ftty cids meets the requirements nd is more ville to the lrve s it is stored in the polr lipid frction (Evjemo & Olsen, 1997; Evjemo et l., 2003; vn der Meeren et l., 2008; Overrein et l., 2010) 1.4 Gut nd liver development As mentioned mrine fish lrve like the lln wrsse re quite smll nd undeveloped t htching (Osse et l., 1997; Yufer & Dris, 2007). The mouth nd nus opens in time for exogenous feeding nd the gut itself is n undifferentited tue with no functionl stomch (Kjørsvik et l. 1991). As the lrve grow the gut will differentite nd will get more nd more developed over time (Yufer & Dris, 2007, Govni et l,. 1986; Wgø et l., 2001). The lln wrsse will never develop functionl stomch (Hmre & Sæle, 2011) nd therefor the functionl development of the gut is importnt for the lrve to e le to digest nd sor the required nutrients for growth nd survivl. The liver is not developed t htching nd ppers t the strt of exogenous feeding. It will grow over time s the fish lrve grows nd ecome more nd more developed (Hoehne- Reitn & Kjørsvik, 2004; Kjørsvik et l., 2004; Young et l., 2006). The liver ctolizes ft, crohydrtes nd protein nd stores energy like glycogen nd lipids (Lie et l., 1986; Young et l., 2006). The liver is esily ffected y mlnutrition or strvtion nd is good indictor of the nutritionl qulity of live feed (Hoehne-Reitn & Kjørsvik, 2004). 3

10 1.5 Aim of the study: The im of this study is to compre two different live feed progrms to see if using intensively rered copepods s live feed for lln wrsse lrve will give etter growth nd development thn the more trditionl live feed of enriched rotifers nd rtemi. We will hve two groups of three replics ech were one group is fed enriched rotifers nd then rtemi, while the other group is fed intensively rered copepods of incresing size exclusively throughout the experiment. To mesure ny difference etween the two groups we re looking t different growth prmeters s stndrd length, myotome height, dry weight nd dily weight increse. In terms of lrvl development we re going to look t the liver nd gut development of the two groups to see if the difference in nutritionl qulity of the live feed will give different development in the limentry system. In the liver nd gut we will look t re size of nuclei nd mitochondri, the re frction of nuclei, mitochondri nd glycogen, the generl ppernce of the liver nd gut tissue nd the ppernce of mitochondril memrnes nd criste. We will lso look t the microvilli density nd height in the gut. 4

11 2. Mterils nd methods 2.1 First feeding experiment. The first feeding experiment ws performed on lln wrsse lrve (Lrus ergylt) nd ws conducted in the CodTech l t NTNU Sel in Trondheim, Norwy. The experiment consisted of two different feeding regimes with three replictes of ech tretment. The first group (the Copepod-group) ws fed Acrti tons exclusively throughout the whole experiment. Strting with nuplii until 15 dys post htch (dph) then s the lrve grew they were fed copepodits from 15 until 30 dph. From 30 to 45 dph the lrve were fed comintion of copepodits nd dult Acrti Tons in 2:1 rtio. The other group (the Rotifer-group) were fed rotifers (Brchionus Sp.) enriched on Multigin (BioMr, Norwy) until 40 dph. From 30 to 40 dph the Rot group ws co-fed enriched rotifers nd enriched Brine Shrimp (Artemi frnciscn) efore the lrve were fed Brine shrimp exclusively until the end of the experiment. Blln wrsse lrve (Lrus ergylt) from Nofim (Sunndlsør, Norwy) ws plced in holding tnk with gentle ertion for cclimtiztion (12 C, 250 L) when they were 2 dph. The following dy (3 dph) the lrvl density ws mesured to get n estimted count of numer of lrve. The lrve were trnsferred to six 100 L conicl ottomed, cylindricl tnks with n estimted numer of 8200 lrve per tnk. From 2 dph to 4 dph the lrve were kept in drkness nd from 4 dph nd throughout the experiment the lrve were kept in constnt illumintion (MASTER TL-D 90 Grphic, 18W/965, Philips, Netherlnds). Temperture nd oxygen levels were mesured every dy (ProODO Opticl Dissolved Oxygen Meter, YSI Inc, OH, USA), eing kept t C (tle 2.1) nd 80 % sturtion respectively. The sewter tht ws pumped into the tnks, ws filtered through sndfilter nd through 1µm mesh to get rid of prticles (Skjermo et l. 1997), nd ws microilly mtured to stilize the cteril content. The wter exchnge ws grdully incresed from 2 to 8 times dy through the experiment. Cermic cly (Vingerling K148, Sielco, Germny) ws dded to the wter to increse the visul contrst nd reduce the cteril lod. With incresing wter exchnge the mount of cly dded to the wter ws incresed from 1.6 g per feeding t the eginning of the experiment to 3 g per feeding t the end of the experiment (tle 2.1). 5

12 Tle 2.1: Experimentl setup nd prmeters for the strt feeding experiment of lln wrsse lrve. Dys post htch Temperture ( C) Dy degrees ## Cly (g/ tnk /dy) - 3,2 4,8 7,5 8 8,5 9 Wter exchnge rte Feeding frequency 2 times dy 3 times dy Rotifer-group: 3 tnks Individuls per feeding - Individuls per feeding Enriched rotifers Enriched rtemi 3000 Copepod-group 3 tnks Copepods Individuls per feeding Stge n c n c n c 67 % c + 33 % 67 % n + 33 % N:nuplii, c:copepodits, : dult 6

13 2.2 Live feed production Cultivtion nd hrvesting of microlge (Rhodomons ltic) Rhodomons ltic (Clone NIVA 5/9 Cryptophyce: Pyrenomondles) were grown in 160 nd 200 litre plexigls cylinders. In periods of incresed production 300 litre plstic gs were lso used. The ph in the cylinders ws kept etween 7.5 nd 8.5 (ph/mv-meter, WTW ph 315i, Germny) nd the culture ws continuously erted with 1-2% CO 2. When overgrowing of the cylinder wlls ws oserved, the tnks were drined of lge nd clened with hot wter nd chlorine, nd then rinsed. To replenish the clened cylinder 50 % of the lge content of neighoring tnk ws trnsferred to the clen tnk, the cylinder filled to the top with sewter nd Conwy medium (1 ml per liter sewter) ws dded. The wter used in the lge cultivtion ws filtered through snd filter, heted to 20 C, nd filtered through 1 µm mesh, efore eing chlorinted (10-15% NOCl, 0.25 ml per litre, no ertion, >5 hours) nd dechlorinted (N 2 S 2 O 3, 3 g per 100 litre, ertion, >5 hours). 50 % of the lge in cylinder were hrvested dily. The remining culture ws diluted with sewter nd 1 ml Conwy medium per liter sewter ws dded Cultivtion of copepods (Acrti tons) Egg production Acrti tons (clone DFH.AT1) ws cultivted under constnt illumintion in three 1700 liter tnks, with flow through of sewter (snd filtered, heted to 20 C nd filtered through 1µm mesh) t wter exchnge rte of once per dy. The sieve covering the wter outlet ws clened every morning to keep the sieve from ecoming clogged. Temperture, O 2 levels, ph nd slinity ws mesured every morning (ProODO Opticl Dissolved Oxygen Meter, YSI Inc., OH, USA; LH-T28, Lohnd, Chin) nd kept t C, over 4 mg L-1 dissolved O 2, nd ppt respectively. Hrvested lge (Rhodomos ltic) were trnsferred to 1000 L tnk nd were fed continuously (Nesse, 2010) to the copepod tnks y n electromgnetic dosing pump (AXS602, Seko, Itly). The copepod eggs were collected dily y siphoning long the ottom of the tnks. The wter collected ws filtered through two sieves with 120 µm nd 100 µm mesh to rinse out ny wste, efore the wter ws filtered through 64 µm sieve to concentrte the eggs. The eggs were rinsed with sewter efore eing stored in sewter in NUNC EsyFlsks (Nunc A/S, Denmrk.) t 2 C. 7

14 Production of copepod nuplii for feed The mount of eggs needed ws emptied from NUNC flsk into 100 L white plstic tnks nd ws kept under moderte ertion nd t C. After 24 h when the eggs htched the nuplii were fed Rhodomons 3 times dy until hrvesting. When collecting the nuplii needed for feeding, the density ws estimted y tking out wter smple from the tnk, fixting the smple with Lugol`s solution nd tking out 7 drops of 50 µl. The individuls in ech drop were counted, the highest nd the lowest count ws cncelled nd the density ws estimted y clculting the men of the 5 remining drops. The desired mount ws hrvested nd concentrted y 64 µm sieve nd stored in 100 L plstic tnk under moderte ertion nd 8 C until they were fed to the lrve(>12 hours). Before feeding the nuplii density in the holding tnk ws estimted, the desired mount for one feeding ws tken out nd concentrted efore eing trnsferred to the lrvl tnks Cultivtion, enrichment nd hrvesting of rotifers (Brchionus sp.) Brchionus sp. ws cultured in three 250 L tnks with conicl ottoms in sewter t 34 ppt nd temperture of C. The rotifers were fed Bker`s yest (2.6 g per million rotifers dy -1 ) dissolved in sewter tht ws continuously pumped into the tnks. For long term enrichment Multigin (0.14 g per million rotifers) lended with sewter ws dded to the rotifer tnks twice dy. Deris ws removed from the tnk dily y flushing from n outlet t the ottom of the tnk for 5 seconds. Due to high mount of prticles in the wter (yest, Multigin, wstes) the rotifer tnks hd to e clened once week. The rotifers were trnsferred into holding tnk while the rotifer tnks were clened out. The tnks were filled with clen wter nd the rotifers were put ck in. The density nd egg rtio of ech culture ws estimted every morning. From wter smples tken from ech tnk 12 drops of 25 µl ws tken out nd the numer of individuls nd numer of eggs were counted in ech drop. The highest nd lowest count ws cncelled out nd the mount in the remining 10 drops ws dded together to get the egg rtio nd further multiplied to get the numer of individuls per liter. The desired mount of rotifers were then tken out from tnk nd concentrted nd wshed in 64 µm sieve. The rotifers were then put in 100 L tnk with sewter (24 C nd moderte ertion) nd were short term enriched with Multigin (0.14 g per million 8

15 individuls) for 2 hours. Before feeding them to the lrve the density ws doule checked efore the desired mount for one feeding ws tken out, wshed nd concentrted. The remining rotifers were stored cold (8 C nd under moderte ertion) to keep the nutritionl vlue stle until the following feedings Cultivtion of Artemi frnciscn Dry Artemi cysts were hydrted in fresh wter (4.9 liter of wter for g cysts, C) under hevy ertion for one hour efore eing decpsulted. The decpsultion process ws done ccording to Sorgeloss et l. (1977) g NOH dissolved in 150 ml of wter nd 1.44 L of NOCl ws dded to the hydrted cysts. The process ws stopped when the cysts chnged color from rown to ornge. The decpsulted cysts were filtered through 125 µm mesh net nd rinsed under running wter for out 5 minutes. The cysts were then concentrted nd stored t 5 C nd used within 6 dys. The desired mount of decpsulted cysts ws put in tnk with 60 L of sewter (25-28 C) with hevy ertion for 24 hours for htching. The htched Artemi ws wshed in n Artemi-wsher for 10 minutes. The wshed nuplii ws then concentrted nd trnsferred to new tnk. The Artemi ws enriched twice with Multigin (10 g/60l; Biomr) during the next 24 hours. The Artemi ws then hrvested, rinsed nd concentrted efore eing fed to the fish lrve. 2.3 Lrvl smpling nd fixtion Fish lrve were rndomly smpled from the tnks using ldle. The ldle ws rinsed with hot fresh wter etween ech smpling to void trnsferring cteri. The first smpling done on 3 dph ws done efore the lrve were trnsferred to the different tretment tnks. After this point n equl numer of individuls were smpled from ech tnk on smple dys. The collected lrve were nesthetized using tricine methnesulfonte (MS-222, Finquel, Agent Chemicl Lortories Inc., USA) nd then rinsed in distilled wter. The fish smpled for histologicl nlysis were fixted in 4 % prformldehyde (PFA) in 0.11M hepes uffer nd stored cold (4 C) in glss vils. 9

16 2.3.1 Stndrd length nd myotome height Mesurement on stndrd length (SL) nd myotome height (MH) were done on lrve collected on 6 different dys: 3, 8, 15, 28, 36 nd 45 dph. On 3 dph 20 lrve were smpled from the egg incutor efore the remining lrve were distriuted to the different tretment tnks. On the other dys lrve were smpled from ech tnk. The lrve were nesthetized nd SL nd MH were mesured from pictures y using Nikon SMZ1000 stereomicroscope nd Infinity 1-3C cmer. SL ws mesured from the tip of the mouth to the end of the notochord on smll lrve nd to the strt of the til fin on lrger lrve. MH ws mesured perpendiculr to the xil skeleton, right ehind the nus. SL nd MH mesurements were performed y Stine Wiorg Dhle (SINTEF). After imge nlysis the sme lrve were rinsed in distilled wter nd smpled for CN-nlysis Dry weight nd dily weight increse The sme lrve used for SL nd MH mesurements were used for CN-nlysis. The lrve were nlyzed for cron content y using n Elementl Comustion Anlyzer (Costech Anlyticl Technologies Inc., USA) with cetnilide s stndrd (nlysis done y Mrthe Schei, SINTEF). Lrvl dry weight ws clculted y using the eqution: DW= (µg cron lrve -1 )* 2.34 (Reitn et l., 1993) Specific growth rte (SGR) nd dily weight increse (%DWI) ws further clculted using these equtions ccording to Ricker (1958): SGR = ln W 2 lnw 1 t 2 t1 %DWI = (e SGR 1) * 100 where W 1 nd W 2 re the dry weight t time t 1 nd t Survivl From 15 dph ded fish lrve were siphoned off the ottom of the tnks nd counted every second/third dy. At the end of the experiment (45 dph) the remining fish in the tnks were counted. The dt ws corrected for smpled lrve nd the lrve tht were ccidentlly removed during clening of the tnks. The mortlity up to 15 dph ws considered to show negtive liner regression with time. 10

17 2.4 Histologicl nlysis 5 fixted lrve from 3 dph nd 5 fixted lrve from ech tretment from 6, 8 nd 15 dph were emedded in EPON (Appendix 1). Sections were mde through the liver nd the midgut (Figure 2.1) for further nlysis in light microscope (1 µm thick sections) nd in trnsmission electron microscope (70 nm thick sections) with Leic Richert Ultrcut microtome (Leic Microsystems, Germny). The 70 nm thick sections for electron microscopy were performed y Tor Brdl, NTNU. Figure 2.1: Blln wrsse lrve 8 dph showing where the liver nd gut sectioning were performed. Photo: Mren Rnheim Ggnt 11

18 2.4.1 Light microscope The sections were stined with Toluidine Blue nd Borte uffer nd studied under Zeiss Axioscope2 plus microscope (Zeiss Inc., Germny) equipped with JVC TK-C1381 color video cmer (JVC, Jpn). The sections were nlyzed with n imge processing progrm clled Imge-J. On oth the sections from the liver nd the gut the nucleus re size ws mesured using Imge J Trnsmission electron microscope The sections were contrsted with led citrte nd oserved in Jeol JEM-1011 trnsmission electron microscope (Jeol LTD, Jpn) t n ccelerting voltge t 80 kv y Tor Brdl (NTNU). The photos tken from the trnsmission electron microscope ws further nlyzed with the imge processing progrm Imge J. In Imge J point grid ws locted on rndomly selected photos (x mgnifiction) tken of the liver nd gut sections. Ech point covered given re. On the liver sections the totl re covered y mitochondri, lipid, glycogen, endoplsmic reticulum (ER), nucleus nd generl cytosol ws mesured. On the photos of the gut sections the re covered y mitochondri, ER, nucleus, generl cytosol nd vcuole 1 nd 2 ws mesured. This gve two-dimensionl quntifiction nd the re frction of the different orgnelles ws clculted. On the photos of the liver nd gut sections mitochondri size ws mesured nd qulittive study on mitochondril structure nd ppernce ws done. On the sections of the gut microvilli height nd density ws lso mesured. 2.5 Sttisticl nlysis Dt tht consisted of percentge vlues were trnsformed using n rc-sine trnsformtion efore eing sttisticlly tested. Difference of mens ws tested using one-wy ANOVA (P<0.05). An F-test were performed to check for homogeneity of vrince (p=0.05). A T-test for equl vrince ws performed if there ws homogeneity of vrince nd T-test for unequl vrince if there ws not. The sttisticl testing ws done in Microsoft Office Excel nd grphs were mde in SigmPlot 12.5 (Systd Softwre Inc., USA 2013). 12

19 3 Results 3.1 Growth nd survivl Dry weight nd dily weight increse Men dry weight (DW) of the lln wrsse lrve t the eginning of the experiment ws ± 2.07 µg. At 8 dph there ws lredy difference in DW etween the lrvl groups, nd t 15 dph the Copepod-group hd significntly higher DW of 191 ± 11 µg lrv -1 versus 82 ± 0.69 µg lrv -1 for the Rotifer-group (Figure 3.1). The difference etween the Rotifer nd the Copepod-group continued pst 28 dph (figure 3.2), ut fter 30 dph, when the Rotifergroup were wened on to Artemi sp., the difference decresed nd there ws no significnt difference etween the groups from 36 dph until the end of the experiment. Both groups hd slowdown in the increse of dry weight etween 28 nd 36 dph, with it incresing gin etween 36 nd 45 dph. For the Rotifer-group this ws the eginning of the wening period on to rtemi which could men tht they hd hrd time ccepting the new feed t first. The slowdown in dry weight increse in the Copepod-group could e due to su-optiml numers nd size of the copepods they were fed. When the feeding ws chnged from copepodits to copepodits nd 1/3 dult copepods t 36 dph the men dry weight incresed more rpidly gin. The dily weight increse (DWI) of oth lrvl groups ws negtive in the first period of the experiment prior to exogenous feeding (Figure 3.3). In the period of 8-15 dph oth groups hd positive DWI with significntly higher DWI in the Copepod-group with 18.8 ± 2.5 % versus 8.7 ± 2 % in the Rotifer-group. Over the next two time intervls, nd dph there ws decrese in DWI for the Copepod-group efore it incresed gin t dph. This cn e explined y the issue of the su-optiml feeding regime discussed erlier. When the dult copepods were introduced in the diet t 36 dph, the DWI incresed gin. The decrese in DWI in the Rotifer-group t dph cn e explined y the wening onto Artemi in this time period. After the lrve were fully wened onto rtemi the DWI incresed gin t dph, nd ws t the sme level s the Copepod-group t round 15 %. Looking t the men DWI for the whole strt feeding period (3-45 dph) there ws no significnt difference etween the groups nd it ws t 10.5 % for the Copeopod-group nd 9.25 % for the Rotifer-group. 13

20 1,00 COP ROT Men dry weigt (mg/lrv) 0,10 0, Dys post htch Figure 3.1: Men dry weight (mg lrv 1 ) of lln wrsse lrv from 3 to 15 dph were Rotifer group were fed rotifers nd Copepod-group fed copepod nuplii (y-xis presented in logrithmic scle). Dt re men ±SE (n=20-35). Letters indictes significnt difference. 10 N C C+A(2:1) Men dry weight (mg/lrv) 1 0,1 COP ROT 0, Dys post htch Figure 3. 1: Men dry weight (mg lrv -1 ) for the whole strt feeding experiment of 45 dys (y-xis presented in logrithmic scle). Letters indictes significnt difference etween the groups. Error rs indictes ± SE. The re etween the dshed lines mrks the time of co-feeding with rotifers nd rtemi (30-41 dph). The rrow heds mrks the time of feeding with copepod nuplii (N) 3-15 dph, copepodits (C) dph nd copepodits nd dult copepods (A) dph. 14

21 25 20 COP ROT c c c Men % DWI c c c c Dys post htch Figure 3.2: Dily weight increse (DWI, %) for the lln wrsse lrve during the whole strt-feeding experiment consisting of 45 dys split into five intervls nd one for the whole experiment. Letters indictes significnt difference etween tretments nd within tretments. Error rs indicte ± SE. 15

22 3.1.2 Stndrd length (SL) nd myotome height (MH) In the eginning of the experiment from 3 to 8 dph there ws negligile increse in SL in oth lrvl groups, nd no significnt difference etween the groups, 3.93±0.07 for the Copepod-group nd 3.87±0.02 for the Rotifer-group (Figure 3.4). From 15 dph nd throughout the experiment the Copepod-group hd significntly higher SL thn the Rotifergroup. The myotome height of the Copepod lrve ws significntly higher thn tht of the lrve from the Rotifer-group t 8 dph nd throughout the whole experiment (Figure 3.5). The Rotifer-group didn t hve significnt increse in myotome height efore 28 dph. 14 Men stndrd lenght (mm) dph COP ROT Dys post htch Figure 3.3: Mesured stndrd lenght (SL) t 3 dph, 8 dph, 15 dph, 28 dph, 36 dph nd 45 dph (n=12 for 3 nd 8 dph, n=20 for 15, 28, 36 nd 45 dph). Letters indictes significnt difference etween tretments. Error rs indicte ± SE. 16

23 Men myotome height (mm) 3,0 2,5 2,0 1,5 1,0 0,5 3 dph COP ROT 0, Dys post htch Figure 3.4: Mesured myotome height (MH) t 3 dph, 8 dph, 15 dph, 28 dph, 36 dph nd 45 dph (n=12 for 3 nd 8 dph, n=20 for 15, 28, 36 nd 45 dph). Letters indicte significnt differences etween tretments. Error rs indictes ± SE Survivl The highest mortlity ws t the eginning of the first feeding experiment. From 15 dph nd throughout the experiment there ws no significnt difference in % survivl etween the two groups. At 45 dph when the experiment ws terminted, the Copepod-group hd slightly higher survivl thn the Rotifer-group, ut the difference ws not significnt. Tle 3.1: Men survivl (%) ± SE of ech tretment t 15, 22, 29, 37 nd 45 dph (n=3). No significnt difference ws found etween the two tretments Men survivl (%) ± SE DPH ROT COP ±1 15 ± ±0 13 ± ±1 12 ± ±1 12 ± ±1 11 ±2 17

24 3.2 Liver morphology Light microscopy The liver tissue in the lrve t 3 dph seemed quite undeveloped with irregulrly shped nuclei without clerly defined nucleoli. In ll of the lrve tested there ws residue of yolk nd syncytium round the liver tissue, nd in two of the lrve tested liver tissue ws not found t ll. At 8 dph the liver tissue ws more developed nd orgnized s loules with visile lood vessels nd lood cells (Figure 3.6 B-C). The nuclei hd rounder shpe with clerly defined nucleoli. The liver in the Rotifer-group t 8 dph seemed less developed with less liver tissue compred to the Copepod-group. At 15 dph the liver tissue in oth groups ws more developed compred to the erlier smple dtes. The liver ws lrger in volume with visile lood vessels nd glycogen deposits throughout the tissue (Figure 3.6 D-E). Figure 3.6 A: Photo microgrph of the liver t 3 dph. The tissue is undeveloped with no visile lood vessels nd irregulrly shped nuclei without defined nucleoli. 18

25 Figure 3.6 B: Photo microgrph of the liver of lrve t 8 dph from the Rotifer-group. The liver is orgnized into loules ut the tissue seems more immture thn the liver tissue in the Copepod-group. The nuclei re rounder in shpe nd hve defined nucleoli. Figure 3.6 C: Photo microgrph of the liver of lrve t 8 dph from the Copepod-group. The tissue seems more developed nd orgnized thn t 3 dph nd compred to the liver tissue from the Rotifer-group. There re visile lood vessels nd lood cells. The nuclei re rounder in shpe with defined nucleoli. 19

26 Figure 3.6 D: Photo microgrph of the liver of lrve t 15 dph from the Rotifer-group. The tissue is more developed thn t the erlier smple dtes. There re visile lood vessels nd lood cells, nd visile glycogen deposits. Figure 3.6 E: Photo microgrph of the liver of lrve t 15 dph from the Copepod-group. The tissue is more developed with visile lood vessels. Glycogen deposits re lso visile throughout the tissue. 20

27 3.2.2 Trnsmission electron microscopy The heptocytes were seen s polygonl cells with lrge, sphericl nuclei (Figure 3.7). Visile orgnelles consisted of nuclei, mny mitochondri nd rough endoplsmic reticulum (ER) which were more defined t 15 dph thn t 3 dph. Glycogen ws oserved in the liver lredy t 3 dph, lthough t low mounts. The mount of glycogen in the liver grdully incresed over time nd ws most undnt in the Rotifer-group t 15 dph (Figure 3.7 D). In the lrve from the Rotifer-group t 15 dph the glycogen ws scttered throughout the cells while in the lrve from the Copepod-group the glycogen ws more clustered together. At 3 nd 8 dph the mitochondril memrnes ws generlly more rugged thn t 15 dph with not much difference etween the groups t 8 dph. At 15 dph the Copepod-group hd lrger proportion of smooth outer mitochondril memrnes thn the Rotifer-group. At 3 dph there were much lrger proportion of mitochondri with less dense criste thn t 8 nd 15 dph (Figure 3.7 A). At 15 dph the ER ws much more undnt nd orgnized thn on 3 nd 8 dph nd is seen enveloping the mitochondri (Figure 3.7 D-E). Figure 3.7 A: Microgrph tht shows the generl morphology of liver tissue from lrve t 3 dph. Nuclei (N) nd mitochondri (M) re shown on the photos. The photos show difference in mitochondril ppernce, undnce nd orgniztion of ER nd occurrence of glycogen over time. Mitochondri memrnes re more rugged (rrow) nd criste less dense (str) t 3 dph thn t the lter smple dtes. Endoplsmic reticulum is more undnt nd orgnized t 15 dph nd glycogen ws seen in oth groups from 8 dph. 21

28 Figure 3.7 B: Microgrph tht shows the generl morphology of liver tissue from lrve from the Rotifer tretment t 8 dph. Mitochondri (M) nd ile ducts (BD) re shown on the photos. Rugged memrnes mrked with rrows. Figure 3.7 C: Microgrph tht shows the generl morphology of liver tissue from lrve from the Copepod tretment t 8 dph. Nuclei (N), mitochondri (M) nd glycogen (G) nd endoplsmic reticulum (ER) re shown on the photos. Rugged memrne mrked with rrow. 22

29 Figure 3.7 D: Microgrph tht shows the generl morphology of liver tissue from lrve from the Rotifer tretment t 15 dph. Nuclei (N), mitochondri (M), glycogen (G) nd endoplsmic reticulum (ER) re shown on the photos. Figure 3.7 E: Microgrph tht shows the generl morphology of liver tissue from lrve from the Copepod tretment t 15 dph. Nuclei (N), mitochondri (M), glycogen (G) nd endoplsmic reticulum (ER) re shown on the photos. 23

30 3.2.3 Surfce re frction of heptocyte components Glycogen ws found in the liver lredy t 3 dph, lthough in low levels, 1.2 ± 0.4 %. The surfce re frction of glycogen incresed over time in oth groups nd the highest frction ws found in the Rotifer-group t 15 dph with 38.3 ± 3 % (Figure 3.7). There ws no significnt difference in the surfce re frction of mitochondri etween or within the two tretments over time. The surfce re frction of the nuclei decresed over time for oth tretments nd the nucleus re frction on 3 dph ws significntly higher thn the nucleus re frction t 15 dph. 100 Men surfce re frction(%) c D2 D8 ROT D8 COP D15 ROT D15 COP c c Nucleus Mitochondri Glycogen Other c c Tretment Figure 3.8: Men surfce re frction (%) of heptocyte cell components (nuclei, mitochondri, nd glycogen) on 3 dph, 8 dph nd 15 dph for oth tretments (n=5). Letters indictes significnt difference etween nd within tretments. Error rs indictes ± SE. 24

31 3.2.4 Are size of nucleus nd mitochondri The nucleus re size ws the smllest t 3 dph (91.2 ± 2.2 µm 2 ), ut it is not significntly smller thn the nucleus size in either group t 8 dph (Figure 3.9 A). There ws slight increse in nucleus re size over time ut only significntly for the Copepod-group t 15 dph (118.6 ± 2.6 µm 2 ). The mitochondri were the smllest t 3 dph (0.74 ± 0.07 µm 2 ), nd there ws significnt increse in mitochondri size from 3 dph to 8 dph for oth groups (Figure 3.9 B). The mitochondri size for the Rotifer-group ws significntly higher thn the Copepod-group on 8 dph nd oth groups on 15 dph (2.85 ± 0.9 µm 2 ), ut the high stndrd error indictes gret vrition in the smple. At 15 dph there ws no difference in mitochondri size etween the groups, with size of round 2 ± 0.3 µm 2 for oth groups. Nucleus re size( m 2 ) A Mitochondri size( m 2 ) B c c c dph D8COP D8ROT D15COPD15ROT 0 3 dph D8 ROT D8 COP D15 ROT D15 COP Tretment Tretment Figure 3.9: Nucleus re size (A) nd mitochondri size (B) in the heptocytes of lrve from 3 dph nd from oth tretments t 8 nd 15 dph. Letters indictes significnt difference etween nd within the groups. Error rs indictes ± SE. 25

32 3.2.5 Mitochondril chrcteristics Over time there is n overll decrese in the occurrence of rugged outer mitochondril memrnes. At 3 dph most of the mitochondri hd rugged outer memrne (96.6 ± 3 %). At 8 dph the proportion of smooth outer memrne hd incresed ut still the mjority hd rugged ppernce. At 15 dph there ws significnt difference etween the two groups, where the Copepod-group hd igger proportion of smooth outer memrnes thn the Rotifer-group. Outer mitochondril memrne ppernce (%) Tretment 3 dph 8 dph ROT 8 dph COP 15 dph ROT 15 dph COP c Smooth Rugged Figure 3.10: Men outer mitochondril memrne ppernce (%) in the liver of lrve t 3 dph nd from oth tretments t 8 nd 15 dph (n=5). The outer mitochondril memrne is clssified s either smooth or rugged. There ws significntly higher occurrence of rugged memrne structure t 3 dph thn t the lter smple dys nd the highest occurrence of smooth memrne structure ws found on 15 dph in the Copepod-group. Letters indictes significnt difference etween nd within tretments. Error rs indictes ± ER. 26

33 Over time there ws significnt decrese in the occurrence of less dense criste nd significnt increse in the occurrence of denser criste. At 8 dph oth groups hd significnt increse in the occurrence of denser criste when compred to 3 dph, where the Rotifer-group significntly higher proportion of denser criste thn the Copepod-group. At 15 dph oth groups hd high proportion of dense criste where the Copepod-group hd significntly higher proportion thn the Rotifer-group. Interdigitted criste were found in individuls from the Rotifer-group oth t 8 nd 15 dph with the highest occurrence t 15 dph (14 ± 4 %). 100 d Men criste ppernce (%) c * Less dense Denser Interdigitted 0 3 dph 8 dph ROT8 dph COP15 dph ROT15 dph COP Tretment Figure 3.11: Men criste ppernce (%) in heptocytes of lrve from 3 dph nd from oth tretments t 8 nd 15 dph (n=5). There ws significnt difference etween the tretments t oth 8 nd 15 dph nd significntly less dense criste t 3 dph. Interdigitted criste ws mostly found in the Rotifer-tretment with the highest occurrence t 15 dph (*). Letters indictes significnt difference etween nd within tretments. Error rs indictes ± SE. 27

34 3.3 Gut morphology Light microscopy At 3 dph the digestive trct ws short nd nrrow with thick intestinl epithelium. The enterocytes were long nd nrrow nd the nuclei were olong in shpe with no distinguishle nucleoli (Figure 3.12 A). At 8 dph the digestive trct ws wider thn t 3 dph nd the enterocytes were more distinguishle s columnr cells with round nuclei (Figure 3.12 B-C). The intestinl epithelium in the Copepod-group ws strting to fold more thn the epithelium in the Rotifer-group. Two of the lrve from the Rotifer-group t 8 dph hd thinner intestinl epithelium with less dense (Figure 3.19) nd shorter microvilli (Figure 3.18), exhiiting signs of strvtion. At 15 dph the epithelium ws thicker nd hd more folds thn t 8 dph for oth groups. The microvilli order on the luminl surfce ws longer in the Copepod-group thn the Rotifer-group t 15 dph nd oth groups t 8 dph (Figure 3 18). At the se of the enterocytes in the Rotifer lrve from 15 dph (Figure 3.12 D) numerous lipid droplets were visile. Lipid droplets were lso seen in the enterocytes of the Copepod-group ut they were more dispersed throughout the cells nd not s numerous. Figure 3.12 A: Photo microgrph of the midgut of lrv t 3 dph. The digestive trct is nrrow with thick intestinl epithelium. Visile microvilli order towrds the digestive trct. The enterocytes nd nuclei re long nd nrrow. 28

35 Figure 3.12 B: Photo microgrph of the midgut of lrv t 8 dph from the Rotifer-group. The digestive trct is wider nd the intestinl epithelium is nrrower thn t 3 dph. The cells re more qudrtic in shpe nd the nuclei hve ecome more round. Visile microvilli order towrds the digestive trct. Figure 3.12 C: Photo microgrph of the midgut of lrv t 8 dph from the Copepod-group. The digestive trct is wider thn on 3 dph. The epithelium is more folded thn in the Rotifer-group. Here the nuclei re lso more round thn t 3 dph. Visile microvilli order towrds the digestive trct. 29

36 Figure 3.12 D: Photo microgrph of the midgut of lrv t 15 dph from the Rotifer-group. The intestinl epithelium is thicker nd more folded thn on erlier smple dtes. Numerous lipid droplets re visile t the se of the enterocytes.. Figure 3.12 E: Photo microgrph of the midgut of lrv t 15 dph from the Copepod-group. The intestinl tissue is thicker nd more folded thn on erlier smple dtes. The microvilli order is thicker nd longer thn in the Rotifer group.. 30

37 3.3.2 Trnsmission electron microscopy The intestinl epithelium of the lrve consisted of tll columnr enterocytes with the nuclei t the se of the cells nd order of microvilli t the top. Visile orgnelles were nuclei, mitochondri nd endoplsmic reticulum (ER). The outer mitochondril memrnes were more rugged t 3 dph thn t lter smple dtes (Figure 3.13 A). At 8 dph oth groups hve lrger proportion of rugged thn smooth memrnes ut less so thn t 3 dph. At 15 dph oth groups hve smoother outer mitochondri memrnes with no ig difference etween the groups. There ws no ig difference in criste density over time either within or etween groups, though more incidents of less dense criste t 3 dph thn t lter smple dtes (Figure 3.13 A). At 15 dph the ER ws much more undnt nd orgnized in the enterocytes of the Copepod-group thn for the Rotifer-group nd oth of the groups t 8 dph (Figure 3.13 E). Two unidentified vcuoles were oserved in the enterocytes of the Rotifer-group t 8 nd 15 dph. Vcuole 1 ws round with white droplets inside (Figure 3.13 B), while vcuole 2 hd grey color with drker outer ring. 31

38 Figure 3.13 A: Microgrph tht shows the generl morphology of intestinl tissue in the midgut from lrve t 3 dph. Nuclei (N), mitochondri (M) nd microvilli (MV) re shown on the photos. The photos show difference in mitochondril memrne structure nd criste density over time nd endoplsmic reticulum gets more undnt nd orgnized over time. At 3 dph the outer mitochondril memrnes re more rugged (rrow) nd the criste less dense (*) thn t 15 dph. 32

39 Figure 3.13 B: Microgrph tht shows the generl morphology of intestinl tissue in the midgut from lrve from the Rotifer-tretment t 8 dph. Nuclei (N), mitochondri (M), vcuole 1 (V1) nd microvilli (MV) re shown on the photo. The rrow mrks mitochondri with rugged outer memrne. Figure 3.13 C: Microgrph tht shows the generl morphology of intestinl tissue in the midgut from lrve from the Copepod-tretment t 8 dph. Nuclei (N) nd mitochondri (M) re shown on the photo. 33

40 Figure 3.13 D: Microgrph tht shows the generl morphology of intestinl tissue in the midgut from lrve from the Rotifer-tretment t 15 dph. Nuclei (N), mitochondri (M) re shown on the photo. Figure 3.13 E: Microgrph tht shows the generl morphology of intestinl tissue in the midgut from lrve from the Copepod-tretment t 15 dph. Mitochondri (M) nd endoplsmic reticulum (ER) re shown on the photo. 34

41 3.3.3 Surfce re frctions of enterocyte components The surfce re frction of nuclei ws highest t 3 dph (20.6 ± 2.3 %) nd decresed slightly over time for oth groups ut there ws no significnt difference etween or within tretments (Figure 3.14). The surfce re frction of mitochondri decresed slightly over time nd there ws significnt difference etween 3 dph nd oth tretments on 8 nd 15 dph (Figure 3.14). The surfce re frction of endoplsmic reticulum (ER) in the enterocytes of the Rotifergroup decreses slightly from 3 to 8 dph ut then seems to stilize t round 25 %. In the Copepod-group there lso is decrese in the ER re frction from 3 to 8 dph, ut t 15 dph there is significnt increse in the frction of ER from 23.5 to 34.2 %. Vcuole 1 nd vcuole 2 ws unidentified vcuoles nd were only found in individuls from the Rotifer tretment t 8 nd 15 dph. The highest vlue of vcuole re frction ws found on 8 dph for vcuole 1 (8.4 ± 1 %), which ws significntly higher thn on 15 dph (3.6 ± 1 %). The surfce re frction of vcuole 2 hd slight increse from 8 to 15 dph ut the difference ws not significnt. 100 Men re frction (%) c Nucleus Mitochondri ER Cytosol Vcuole type 1 Vcuole type 2 c 0 D2 D8 ROT D8 COP D15 ROT D15 COP Tretment Figure 3.14: Men surfce re frction (%) of enterocyte cell components (nuclei, mitochondri, endoplsmic reticulum (ER), generl cytosol, vcuole 1 nd 2 nd microvilli) on 3 dph, nd for oth tretments t 8 dph nd 15 dph (n=5). Letters indictes significnt difference etween nd within tretments. Error rs indictes ± SE. 35

42 3.3.4 Are size of nucleus nd mitochondri There ws n overll increse in nucleus re size over time for oth groups. The smllest nucleus re size ws found in the Rotifer-group (89.2 ± 2 µm 2 ) t 8 dph which ws ctully smller thn t 3 dph (102.6 ± 3.4 µm 2 ). The lrgest nucleus re size ws found in the Copepod-group t 15 dph (137.8 ± 3.2 µm 2 ) which ws significntly lrger thn the nuclei t 3 dph nd the nucleus re size in the Rotifer group t 8 dph. The generl trend for mitochondril re size for oth groups ws decrese in size over time ut there ws no significnt difference etween the groups or etween the groups nd 3 dph. The lrgest mitochondril re size ws t 8 dph in the Rotifer-group (1 ± 0.08 µm 2 ) nd the smllest in the Rotifer-group t 15 dph (0.73 ± 0.06 µm 2 ) A c c c 1,2 1,0 B Nucleus Are size( m 2 ) Mitochondril re size ( m 2 ) 0,8 0,6 0,4 20 0,2 0 D3 D8 Rot D8 Cop D15 Rot D15 Cop 0,0 D3 D8 Rot D8 Cop D15 Rot D15 Cop Tretments Tretment Figure 3.15: Nucleus re size (A) nd mitochondri size (B) in the enterocytes of lrve from 3 dph nd from oth tretments t 8 nd 15 dph. Letters indictes significnt difference etween nd within the groups. No significnt difference in mitochondri size ws found. Error rs indictes ± SE. 36

43 3.3.5 Mitochondril ppernce The highest occurrence of rugged outer mitochondril memrnes ws seen t 3 dph nd there seemed to e generl decrese in the occurrence of rugged memrnes over time for oth groups. At 15 dph the lrve from the Copepod-group seemed to hve more mitochondri with smooth outer memrnes thn the lrve from the Rotifer-group, ut the difference etween the groups were negligile. 100 Mitochondril memrne ppernce (%) c c c Smooth Rugged 0 3 dph 8 dph ROT 8 dph COP 15 dph ROT 15 dph COP Tretment Figure 3.16: Men outer mitochondril memrne ppernce (%) in the enterocytes of lrve t 3 dph nd from oth tretments t 8 nd 15 dph (n=5). There ws significntly higher occurrence of rugged memrne structure t 3 dph thn t the lter smple dys nd the highest occurrence of smooth memrne structure ws found on 15 dph in the Copepod-group. Letters indictes significnt difference etween nd within tretments. Error rs indictes ± ER. 37